Irdye 680rd streptavidin

Protein samples were separated on an 8% SDS-PAGE gel and transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked in TBST+5% BSA, washed in TBST, incubated with IRDye 680RD Streptavidin (1:1000, LI-COR) in TBST, washed twice in TBST and washed once in PBS. The membrane was scanned using a LI-COR Odyssey Infrared Imager.

50 µg of the total RNA isolated from SH-SY5Y cells differentiated as above and treated with 1000 μM OT for 72 h were used to synthesize biotin-labeled probes with a pooled set of primers complementary to 205 human cDNAs spotted on the Atlas^TM Human cDNA Apoptosis Array (BD Biosciences Clontech, 7743–1) using the BD SpotLight^TM Labeling Kit (BD Biosciences Clontech, 634801). Probes were detected via labeling with IRDye-680RD-streptavidin (1:7000, Licor Biosciences, 926–68079). Signal visualization was performed using the Odyssey Infrared Imaging System (LICOR Biosciences). Analysis and quantification was conducted using the AtlasImage 2.0 software (BD Biosciences Clontech) and global normalization method (sum method). Genes that showed no expression values in one or more analyzed experimental groups (control vs treatment, n = 3) were excluded from further analysis. The gene expression responses were calculated as ratios between OT and control, and compared to the expression level of housekeeping gene (60 S ribosomal protein (L13A)). The cDNA array data described here have been deposited at the NCBI gene expression and hybridization array data repository (GEO): under Accession No. GSE93842.

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) was performed on purified mNOTCH1 EGF1-18 and hTHBS1 TSR1-3 proteins (~100 ng) to biotinylate proteins modified with 6- or 7-Alk-Fuc. The reaction was performed with 100 µM Biotin picolyl azide (Click Chemistry Tools, Scottsdale, AZ, USA), 500 µM BTTP (3-(4-((bis((1-tertbutyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)-propan-1-ol, a gift from Dr. Peng Wu) [62 (link)], 250 µM copper sulfate, 2.5 mM sodium ascorbate in water and gently shaking at room temperature for one hour. The biotinylated samples were then analyzed by Western blot. Samples were loaded onto 4–15% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-Myc antibody (Clone 9E10, Invitrogen, 1:2000), followed by IRDye 680-conjugated goat anti-mouse IgG antibody (LI-COR, 1:5000) to detect the total protein amount. To detect the biotinylated proteins, the nitrocellulose membrane was probed with IRDye 680RD streptavidin (LI-COR, 1:2000) or IRDye 800CW streptavidin (LI-COR, 1:2000). The Western blot bands were visualized using an Odyssey System (LI-COR).

Monoclonal antibodies used herein included anti-FLAG (Sigma), anti-HA (BioLegend anti-HA.11), anti-HIV capsid p24 (183-H12-5C, NIH AIDS Research and Reference Reagent Program), anti-Ki67 (abcam, ab16667), anti-tubulin (Sigma, T9026), and anti-V5 (Invitrogen, R960-25). Polyclonal antibodies were purchased from ThermoFisher (anti-V5, PA1-993), or Abcam (anti-CC137, ab185368 or ab183864; anti-gamma H2AX phosphor S139, ab11174; anti-Fibrillarin, ab5821). Secondary antibodies included goat anti-mouse or anti rabbit IgG conjugated to Alexa Fluor 488 or Alexa Fluor 568 (Invitrogen) for immunostaining, or IRDye 800CW and IRDye 680, or IRDye 680RD Streptavidin (LI-COR Biosciences) for Western blot analysis.